The present invention relates to a method of manufacturing biochips. More particularly, the present invention relates to a method of manufacturing biochips, such as a DNA microarray, which enables the operation of densely aligning and fixing droplets with a minute volume onto a predetermined substrate (micro spot-forming operation) to be performed with increased precision, thereby shortening the time required for the micro spot-forming operation.
Progress in methods for analyzing gene structures has been remarkable in recent years. A number of gene structures, including structures of human genes, have been identified. Biochips such as a DNA microarray in which micro spots containing several thousands to several tens of thousands different DNA fragments are arrayed on a substrate, such as a glass microscope slide, are used in the analysis of such gene structures.
A quill method, pin-and-ring method, and spring pin method have been widely used for forming micro spots in the preparation of DNA microarrays and the like. In all these methods it is necessary to suppress fluctuations in the volume and shape of the micro spots, to maintain a fixed distance between the micro spots, and to prevent contamination due to mutual invasion of the micro spots. In view of higher density requirements in the future, increased precision and speed in the micro spotting operation and improvement in the resulting product quality are desired.
In the quill method, a sample stored in a recess formed at the tip of a pin is transferred onto a substrate by causing the tip to come in contact with the substrate to form a micro spot. The pin tip may be deformed or damaged by contact with the substrate, causing a durability problem. Another problem is cross contamination due to incomplete washing of the sample stored in the recess.
In the pin-and-ring method, a sample solution in a microplate is reserved in a ring. A pin tip dispenses the solution so that the solution reserved goes through the inside of a ring and forms spots on a substrate. The number of kinds of samples capable of reserving in one operation depends on the number of rings. Because that number is conventionally several at most, in order to form several thousand to several tens of thousands kinds of micro spots, about several hundred to several thousand times of washing and drying operations are required. This method has a problem in the productivity.
The spring pin method is a method of producing micro spots on a substrate by transferring a sample attached to a pin tip by pressing the pin tip against the substrate. The device has a double pin structure enclosed in a spring to avoid damage to the pin and substrate when the sample is dispensed. This method, however, can basically spot only one spot per each reserving. Thus, this method also have a problem in the productivity.
To improve productivity, a method of placing a large number of substrates of several tens of sheets on a manufacturing apparatus at one time and forming micro spots may be a possible idea. This method, however, requires a large-scale manufacturing facility and involves a high cost. Even if biochips are manufactured using a large unit, the time required for processing one sheet of substrate (i.e. the time for discharging a sample on one substrate and forming spots) is increased, resulting in a lengthened period of time for completing the process for all substrates. Therefore, the sample used at the initial spotting operation and that at the final spotting operation may not be the same due to denaturing, and this causes a problem that the quality of the resultant biochip is deteriorated. The problem is particularly serious in the case of biological substances which are easily denatured.
In another method for manufacturing a large number of substrates of several tens of sheets, a plurality of units are used to perform the above-described spotting operation side by side at the same time. This method not only involves a high equipment cost, but also requires samples for spotting to a number of units, posing a problem when the sample is a biological substance which is precious and available only in a small amount. Such a method, even if materialized, brings about another problem of fluctuation in the quality each time spotting rods are replaced for a number of units.
Spotting with the application of an ink jet method used in ink jet printers has also been studied. For example, there is disclosed that an ink-jet recording head, in which the ink-discharge nozzle hole is designed to have at least one corner to provide a capillary force (JP-A-59-178258).
There is disclosed an ink jet head in which the sample discharge port has a symmetrical 2n-polygon (n=3 or more) and the cross-section of the ink flow path in the direction vertical to the ink discharge direction has a trapezoidal configuration (JP-A-3-101960).
The manufacturing method using such an ink-jet system excels in an increased speed of spotting operation and uniform spotting product quality.
In the manufacturing method using such an ink-jet system, however, samples are sent to discharge heads to discharge micro liquid spots directly from the outside via a thin tube connected to the head. Because some samples may adhere to the wall of this thin tube, the amount of samples required increases as the volume of the tube increases. Thus, the precious sample is unavoidably wasted.
In addition, a longer time is required for thoroughly washing the tube when discharge heads are washed before charging different samples.
Furthermore, a longer time is required for removing the discharge heads from the unit because of the connected tube. Since frequent removal of once installed discharge heads is impractical, operations for washing the discharge heads and charging and discharging the samples are carried out with the discharge heads installed. Therefore, the washing operation involves a number of physical restrictions, resulting in insufficient washing.
Moreover, confirmation of discharging is difficult because discharging must be confirmed with the discharge heads as installed on the unit. If any discharge head malfunctions, discharging of that part of the unit must be suspended. Otherwise, it is impossible to operate the unit and start the spotting operation until the time when the discharging is restored and normal discharging can be completely confirmed.
If the unit is restarted without complete confirmation of normal discharging or if discharging operation is continued with bubbles and the like lingering in the tube, discharging becomes unstable while the spotting operation is continued on a number of substrates. Some substrates may have spots dropped on deviated points or may not have required spots on proper points, resulting in impaired quality of biochips.
For example, many biological samples containing a DNA and the like have a high viscosity. These samples are required to be rapidly dried upon discharge and attachment to a substrate before spots expand on the substrate. These samples tend to dry or increase the viscosity at a nozzle tip. The nozzle tip may become choked and cannot discharge the sample.
The present invention has been achieved in view of the above problems and has an object of providing a method of manufacturing biochips such as a DNA microarray which enables an operation of densely aligning and fixing droplets with a minute volume on a predetermined substrate (micro spot-forming operation) to be performed with increased precision, thereby shortening the time required for the micro spot-forming operation.